Amelogenins, secreted by ameloblasts, are the predominant proteins in the enamel extracellular matrix, regulating enamel formation. Leucine-rich amelogenin peptide (LRAP) is one of the most abundant alternatively spliced amelogenins. LRAP has been shown to be present in both dental epithelial and mesenchymal tissues, suggesting that it has a role in regulation of odontogenic cell proliferation and/or differentiation. Our preliminary data have shown that LRAP promotes enamel organ epithelial cells to differentiate in vitro. In addition, our results indicated that amelogenin-null mice showed disorganized enamel epithelial cells, characterized by poorly defined stratum intermedium and stellate reticulum, and lacked of secretory ameloblasts without evidence of Tomes'processes and limiting enamel matrix secretion. In contrast, LRAP overexpressor in amelogenin-null (LRAP+/null) background rescued the phenotype of stratum intermedium, stellate reticulum, and restored enamel matrix secretion. However, LRAP+/null mice had a delayed in enamel mineralization, associated with evidence of retained enamel matrix proteins, suggesting that LRAP does not promote mineralization, but only acts as a signaling molecule. In this grant proposal, we will explore the signaling role of LRAP, both in vivo and in vitro. The overall hypothesis of these studies is that LRAP promotes enamel organ epithelial cell differentiation. Our specific aims are as follows: Aim 1:To determine the effect of LRAP on differentiation of enamel organ epithelial cell in vitro. Enamel matrix gene expression, protein synthesis, and in vitro mineral formation by ex vivo human enamel organs and dental epithelial cells treated with exogenous addition of recombinant human LRAP, stably transfected with Tet-regulated promotor within epithelial cell-specific mammalian expression vector containing cDNA encoding for LRAP will be compared to the controls without LRAP intervention. The use of LRAP overexpression in dental epithelial cells will determine the effect of posttranslational modified LRAP on cell differentiation as compared to the unposttranslational modified LRAP from E. coli. The incorporation of Tet- responsive system within mammalian expression vector will allow the regulation of LRAP signaling to promote dental epithelial cell differentiation. Aim 2: To determine the effect of LRAP on enamel epithelial cell differentiation and enamel formation in vivo. Molars and incisors of wild-type, amelogenin-null mice and transgenic mice, including LRAP over- expressors in wild-type (LRAP+/WT) and amelogenin-null (LRAP+/null) backgrounds will be examined under H&E and Trichrome staining for differences in enamel organ cellular morphology and matrix at different stages of development. Alterations in expression of enamel matrix proteins related to enamel epithelial cell differentiation will be analyzed at both mRNA and protein levels. Enamel nanohardness properties of these mice will be compared using atomic force microscopy (AFM), and enamel structural differences will be examined with scanning electron microscopy (SEM) analysis. Public Health Relevance: Tooth regeneration has been gaining significant attention in the recent years from reconstitution studies of epithelial and mesenchymal cells isolated from the developing tooth buds. However, the mechanisms of dental epithelial cell differentiation into functional enamel- forming cells are not well understood. Elucidating the signaling pathways of enamel epithelial cell differentiation is critical for advancement toward bioengineering tooth enamel. In addition, these studies of LRAP signaling may also be applied as a broader model to study the potential multifunctionality of the protein in other cells and tissue types such mesenchymal cells, brain, eye, calvariae, where LRAP expression has also been detected. Our goal is to understand the mechanisms of LRAP signaling to promote dental epithelial cell differentiation. We hypothesize that under the effect of LRAP signaling, enamel organ epithelial cells differentiate into functional enamel-forming cells.